Detection of the presence of microorganisms, their enumeration, identification, and susceptibility to antimicrobic agents are the main goals of a diagnostic microbiologist. The present invention utilizes a method for detecting microbial metabolism that can be applied to each of these areas.
The early detection of bacteria in body fluids (blood, urine, spinal fluid, abscess exudates) is of paramount importance. The usual method for the detection of bacteria in blood is to inoculate 5 ml of the fluid into a culture medium and wait for the appearance of turbidity which is an indication of bacterial growth. Bottles are inspected daily for turbidity or any other changes indicative of microbial growth. While this method is laborious and slow, it allows for the detection of most organisms. Once an organism is detected, a small aliquot of the organism containing culture media is transferred to a petri dish containing appropriate growth media. The subsequent isolated colonies are used for identification and to test for antibiotic susceptibility.
In the mid nineteen seventies, a radiometric technique for the detection of biological activity in blood was developed. In that method, samples of blood are inoculated into a suitable growth medium that includes a C.sup.14 containing carbon and energy source. The inoculated medium is incubated for a suitable period and a portion of the gaseous atmosphere is analyzed for C.sup.14 O.sub.2 (U.S. Pat. No. 3,676,679, issued Jul. 1972). A commercial instrument utilizing this technology is available from Becton Dickinson, Johnston Laboratories, 383 Hillen Road, Towson, Mary. 21204. The disadvantages of the system include hazard due to the necessity to handle radioactive material, environmental contamination resulting from multiple entry of the needle through a septum to the sample,s head space, and lack of total automation. More recently a non-radiometric method for the detection of CO.sup.2 in the gaseous atmosphere was introduced by Becton Dickinson, eliminating the need to handle radioactive material.
An ideal system for the detection of organisms in body fluids should be totally automated (hands-off), quickly detect the presence of microorganisms, and have non-invasive sampling.
The detection and enumeration of microorganisms in industrial samples (foods, cosmetic, water and pharmaceutical samples, water) has followed a different course of technology. The standard method of analyzing samples is the plate count methodology as shown in Standard Methods for the Examination of Water and Water Waste (1985), 7th Ed. APHA, AWWA, WPCF (PP860-866). By this method, a sample is homogenized and diluted with sterile water. Each decimal dilution of 10.sup.-1 to 10.sup.-4 of the homogenate is poured into a petri dish together with a nutrient medium. The dish is incubated for 24 to 48 hours and the number of colonies on the plate are counted. As an alternative, some laboratories now use automated colony counters. In any event, the standard plate count (SPC) method is extremely time consuming, costly and tedious. Additionally, colony forming units do not always correlate with desired safety parameters being estimated.
A wide variety of alternative techniques have been introduced for faster detection and enumeration of microorganisms in industrial samples including: impedance, conductance, turbidity, CO2, ATP determination etc. Impedance and conductance methods measure changes in the electrical properties of the growth media as a result of bacterial metabolism. Both impedance and conductance lend themselves to full automation and result in much faster detection of microorganisms than is possible with the SPC methodology. The limitations of these methodologies include sensitivity of the signal to stray electrical interference, sensitivity to temperature fluctuations, no visible back up to assure that the data is accurate and as a result false positives may be often encountered.
In the turbidity method, the time required to obtain a certain absorbance is recorded. Growth curves are derived . The disadvantages of the system include interference due to turbid material in the samples resulting in the need for dilutions. The method tends to be slower than impedance but faster than SPC.
The ideal system should be fully automated, quickly detect and enumerate microorganisms, and possess a visual backup capability.
The most common techniques for bacterial identification relate to the organism's biochemical properties. Each organism possesses a unique set of enzymes. By performing a series of chemical reactions in growth media, organisms can be identified by a combination of positive and negative reactions that effectively provide a biochemical fingerprinting of the organisms.
Typical identification reactions include carbohydrate fermentation, utilization of substrates such as citrate and urea, production of hydrogen sulfide, indole, lysine decarboxylase etc. or inhibition resulting from antimicrobic agents. A reaction result is determined by a visual color change in the medium or by the presence of turbidity.
The color reagent in most cases is a pH indicator which measures the alkalinity or acidity resulting from the chemical reactions. Another mechanism for color development is the enzymatic splitting of chromogens. A number of manual systems such as API20 (Analytab Products, Plain View, N.Y.), EnterotubeII (Roche Diagnostis, Nutly, NJ), Minitek (BBL Microbiological systems, Cockeysville, MD) are based on this principle.
The positive and negative tests generate a profile number which can be correlated to an organism identification by use of the systems data base. The results are usually available in 12-18 hours and a significant amount of manual manipulation is required.
The trend over the last ten years has been toward automation of these tests to reduce hands on technical time and in some instances to decrease the time for obtaining results. The Autobac IDS system (General Diagnostics, Morris Plains, N.J.) is a semi-automated system and measures microbial growth by light scattering at a fixed 35 angle (U.S. Pat. No. RE28,801). The Automicrobic System (Vitek System, Inc., Hazelwood, Mo.) is a fully automated system (Gibson et al U.S. Pat. No. 3,957,583; Charles et al U.S. Pat. No. 4,118,280; and Charles et al U.S. Pat. No. 4,116,775). The bacterial suspension is drawn into small wells of a card cuvette. The cards are inserted into the machine that monitors changes in optical absorbance. The card is moved automatically into the sensing slot and is monitored every 30 minutes. The Avantage Microbiology Center (Abbott Laboratories, Irving, TX) uses changes in optical density or turbidity. American MicroScan (Baxter Health Care Corp., West Sacramento, Calif.) has an automated system that scans each well of a multi-well tray containing many liquid-based fluorescent assays. A single light source is passed through the wells. Each well's fluorescence is read in sequence. The resulting fluorescence intensity information is transferred to a computer for interpretation using probabalistic methods (U.S. Pat. No. 4,448,534).
The preferred system for the identification of microorganisms should be fully automated, identify quickly (2-3 hours), have a color read visual backup and allow for simple inoculation.
The traditional method used for testing antimicrobial susceptibility has been the standardized disk diffusion method described by Kirby and Bauer (Bauer, Kirby et al. 1966, American J. Clinical Pathol., Vol. 45 (4), p493). According to this method and subsequent modification, colonies are picked and suspended in liquid to result a predetermined turbidity, and streaked onto a nutrient agar in a petri dish. Paper disks impregnated with different anti-microbial material are placed on the inoculated agar surface, and the drug is allowed to diffuse through the agar, forming a gradient around the paper disk. As bacteria grow, they form a visible film on the agar surface. However, around the antibiotic impregnated disks, growth is inhibited if the organism is susceptible to the agent. The zone of the inhibition around the disk is proportional to the degree of susceptibility. The disadvantages of the method are the long incubation time required (18 hours), lack of standardization, and lack of quantitation.
A more common method for antimicrobic susceptibility is minimal inhibitory concentration (MIC). The MIC is determined by making serial dilutions of the drug in a nutrient broth, and inoculating each dilution with a standardized suspension of bacteria. After incubation the various dilutions are examined for turbidity. The MIC is defined as the lowest antibiotic concentration that inhibits macroscopic growth of the test organism. Trays of microtubes are commercially available with frozen or dry solutions of various antibiotics such as those offered by Micro-Media Systems (Potomac, Md.); MicroScan (Baxter Healthcare, W. Sacramento, Calif.); Pasco Laboratories (Wheat Ridge, Co.), Sceptor (BBL, Houston, Tex.). These trays are manually inoculated, incubated for 16-18 hours and often manually read.
The same automated systems used for the identification of microorganisms are also used for MIC detection.
The present invention utilizes fluorescence techniques to achieve a quick, efficient and sensitive means for detecting the presence of microbes, enumerating and identifying microbes, and testing antimicrobial susceptibility of microbes.
Fluorescence is attractive as a detection method due to its inherent sensitivity. The lower limits of detection are in the range of a few thousandths to a tenth of a part per million. A small number of microorganisms can be rapidly detected by means of fluorescence analysis. Koumura et al 1986 (U.S. Pat. No. 4,591,554) used specific umbelliferon derivatives such as 4-methylumbelliferyl phosphate and 4-methyl umbelliferyl galactoside to determine the sanitary quality of various kinds of foods, beverages, water and toilet articles. Sensititer (Gibco Laboratories, Andover, MA) uses fluorescent substrates (such as umbelliferons and coumarin drivatives) that are non-fluorescent until acted on by microorganisms. American MicroScan (Baxter Health Care Corp., West Sacramento, Calif.) uses a series of fluorogenic substrates and indicators consisting of 4-methylumbelliferyl compounds and 7-amido-4-methylcoumarin compounds to rapidly identify microorganisms (2-4 hours) and for rapid determination of their antimicrobic patterns (6-8 hours).
The U.S. Pat. No. 4,495,293 to Shaffar, issued Jan. 22, 1985 discloses a method for fluorometrically determining a ligand in an assay solution wherein the intensity of the fluorescence emitted by the assay solution is related to the change in the transmittance properties produced by the interaction of the ligand to be determined and a reagent system capable of producing a change in the transmittance properties of the assay solution in the presence of the ligand. This is an example of a two dye system wherein one dye in the reagent system interacts with a specific ligand. The interacting dye has an optical spectrum which changes upon interaction with the ligand thereby effecting the fluorescence emission from a second dye. The process thereby measures the presence and amount of the ligand in the sample at dye concentrations that may be toxic to life forms but are effective at measuring ligands.
The present invention utilizes a two dye fluorescence emission system which does not detect or measure a ligand but rather allows or detects microbial growth and utilizes this information for the detection, enumeration, identification, and susceptibility testing of the microorganisms.